C M J a b c a A A K C B S N H 1 m e ( w o e ( e b o t o C 1 d Respiratory Physiology & Neurobiology 179 (2011) 227– 234 Contents lists available at SciVerse ScienceDirect Respiratory Physiology & Neurobiology j our nal ho me p age: www.elsev ier .com/ locate / resphys io l hemosensory control by commissural nucleus of the solitary tract in rats ichele T. Faveroa,1, Ana C. Takakurab,1, Patrícia M. de Paulaa, Eduardo Colombaria, osé V. Menania, Thiago S. Moreirac,∗ Department of Physiology and Pathology, School of Dentistry, São Paulo State University (UNESP), 14801-903 Araraquara, SP, Brazil Department of Pharmacology, Institute of Biomedical Science, University of São Paulo (USP), 05508-900 São Paulo, SP, Brazil Department of Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo (USP), Av. Prof Lineu Prestes, 1524, 05508-900 São Paulo, SP, Brazil r t i c l e i n f o rticle history: ccepted 18 August 2011 eywords: entral and peripheral chemoreflex reathing ympathetic activity TS ypoxia a b s t r a c t The commissural nucleus of the solitary tract (commNTS) is a main area that receives afferent signals involved in the cardiovascular and respiratory control like those related to chemoreceptor activation, however, the importance of the commNTS for the cardiorespiratory responses to chemoreceptor activa- tion is still controversial. In the present study, we investigated the cardiorespiratory responses to hypoxia or hypercapnia in anesthetized and conscious rats treated with injections of the GABA-A agonist musci- mol into the caudal portion of the commNTS. Male Holtzman rats (280–300 g) were used. In conscious rats that had a stainless steel cannula previously implanted into the commNTS, the injection of mus- cimol (2 mM) into the commNTS reduced the pressor response (16 ± 2 mmHg, vs. saline: 36 ± 3 mmHg) and the increase in ventilation (250 ± 17 ml/min/kg, vs. saline: 641 ± 28 ml/min/kg) produced by hypoxia (8–10% O2). In urethane anesthetized rats, the injection of muscimol into the commNTS eliminated the pressor response (5 ± 2 mmHg, vs. saline: 26 ± 5 mmHg) and the increase in phrenic nerve discharge (PND) (20 ± 6%, vs. saline: 149 ± 15%) and reduced the increase in splanchnic sympathetic nerve dis- charge (sSND) (93 ± 15%, vs. saline: 283 ± 19% of baseline) produced by hypoxia. However, muscimol injected into the commNTS did not change hypercapnia (8–10% CO2) induced pressor response or the increase in the sSND or PND in urethane anesthetized rats or the increase in ventilation in conscious rats. The present results suggest that the cardiorespiratory responses to hypoxia are strongly dependent on the caudal portion of the commNTS, however, this area is not involved in the responses to hypercapnia. . Introduction The neural mechanisms involved in the control of breathing ust be responsive to challenges affecting O2, CO2, and pH lev- ls in the body, such as exercise, sleep, hypercapnia and hypoxia Feldman et al., 2003; Nattie, 2006). The physiological process by hich blood gases are detected, called chemoreception, depends n chemical sensors present in the aortic or carotid body (periph- ral chemoreceptors) and within the central nervous system (CNS) central chemoreceptors) (Ballantyne and Scheid, 2001; Feldman t al., 2003; Guyenet, 2008; Loeschcke, 1982). The peripheral chemoreceptors, located mainly in the carotid ody in the rat, detect changes in the partial O2 pressure (PO2 ) r the CO2 pressure (PCO2 ) in the arterial blood and send signals hrough the glossopharyngeal nerve to the commissural region f the nucleus of the solitary tract (commNTS) (Blessing, 1997; ampanucci and Nurse, 2007; Colombari et al., 1996; Finley and ∗ Corresponding author. Tel.: +55 11 3091 7764; fax: +55 11 3091 7285. E-mail address: tmoreira@icb.usp.br (T.S. Moreira). 1 These two authors contributed equally to the study. 569-9048/$ – see front matter Crown Copyright © 2011 Published by Elsevier B.V. All rig oi:10.1016/j.resp.2011.08.010 Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. Katz, 1992; Sapru, 1996; Smith et al., 2006). Similar to the hypoxia, the intravenous (iv) injection of low dose of potassium cyanide (KCN) activates the peripheral chemoreceptors producing tachyp- neic, pressor and bradycardic responses that are abolished by the bilateral ligature of the carotid body arteries (Braga et al., 2007; Franchini and Krieger, 1993; Haibara et al., 1999; Moreira et al., 2006). The pressor and bradycardic responses to i.v. KCN are also abolished by electrolytic lesions of the commNTS (Colombari et al., 1996). Under anesthesia, the activation of breathing and the rise in sympathetic nerve discharge (SND) caused by carotid body stimula- tion are blocked by the injection of glutamatergic antagonists into the commNTS, which suggests that the primary afferent neurons are likely glutamatergic (Sapru, 1996). Detection of an increase in PCO2 by central and peripheral chemoreception acts to maintain stable arterial PCO2 (Feldman et al., 2003; Smith et al., 2006). It is still not clear whether the central chemoreception depends on a few specialized cell clusters located within the brainstem or on multiple types of acid-sensitive neurons (Chernov et al., 2008; Guyenet et al., 2010; Nattie and Li, 2009). The retrotrapezoid nucleus (RTN), locus coeruleus, medullary raphe, hypothalamic orexinergic neurons and the NTS neurons are the main sites suggested to be involved with the hts reserved. dx.doi.org/10.1016/j.resp.2011.08.010 http://www.sciencedirect.com/science/journal/15699048 http://www.elsevier.com/locate/resphysiol mailto:tmoreira@icb.usp.br dx.doi.org/10.1016/j.resp.2011.08.010 2 logy & c D e 2 t N S n r t t r e B a o N ( i n l p ( b 2 2 w s ( t w a I w m 2 2 k o K c m t r m ( r a d s r i x 1 i p 28 M.T. Favero et al. / Respiratory Physio entral chemoreception (Abbott et al., 2009; Biancardi et al., 2008; ean et al., 1989; Deng et al., 2007; Johnson et al., 2008; Moreira t al., 2007; Mulkey et al., 2004; Nattie and Li, 2008; Richerson, 004; Takakura et al., 2006; Williams et al., 2007). The main focus of the present study is to reexamine the ques- ion whether the NTS, particularly the commissural division of the TS caudal to the area postrema, is involved in chemoreception. tudies from the literature have suggested that acid-responsive eurons are located in the NTS and the acidification of the NTS egion alters breathing (Dean et al., 1989; Nattie and Li, 2008). Addi- ionally, previous studies have tested the effects of the lesions or he glutamatergic blockade in the commNTS, suggesting that this egion is essential for the cardiorespiratory responses to the periph- ral chemoreceptor activation (Colombari et al., 1996; Sapru, 1996; raga et al., 2007). On the other hand, a more recent study evalu- ted the effects of muscimol microdialysis in a more rostral portion f the commNTS, suggesting that the rostral portion of the comm- TS is involved mainly with respiratory responses to hypercapnia Nattie and Li, 2008). Based on the assumptions described above, n the present study, also using muscimol injection to block the euronal activity, we investigated the importance of the neurons ocated in a more caudal portion of the commNTS for the cardiores- iratory responses elicited by chemoreflex activation with hypoxia 8–10% O2 in the breathing air) or hypercapnia (8–10% CO2 in the reathing air) in conscious or anesthetized rats. . Methods .1. Animals The experiments were performed on 36 male Holtzman rats eighing 300–330 g. The animals were housed individually in tainless steel cages in a room with controlled temperature 24 ± 2◦ C) and humidity (55 ± 10%). Lights were on from 7:00 am o 7:00 pm. Standard Guabi rat chow (Paulinia, SP, Brazil) and tap ater were available ad libitum. The experimental protocols were pproved by the Animal Experimentation Ethics Committee of the nstitute of Biomedical Science of University of São Paulo. All efforts ere made to minimize animal discomfort and the number of ani- als used. .2. Surgical procedures .2.1. Conscious animals Rats were anesthetized with intraperitoneal (i.p.) injection of etamine (80 mg/kg of body wt) combined with xylazine (7 mg/kg f body wt) and placed in a stereotaxic frame (model 1760; David opf Instruments). A stainless steel cannula was implanted into the ommNTS using the coordinates: 15.0 mm caudal to bregma, in the idline and 7.5 mm below dura mater. The cannulas were fixed o the cranium using dental acrylic resin and jeweler screws. Rats eceived a prophylactic dose of penicillin (30,000 IU) given intra- uscularly and a subcutaneous injection of the analgesic Ketoflex ketoprofen 1%, 0.03 ml/rat) post-surgically. After the surgery, the ats were maintained in individual box with free access of tap water nd food pellets [Guabi rat chow (Paulínia, SP, Brazil)] for at least 7 ays before the tests. To record pulsatile arterial pressure (PAP), mean arterial pres- ure (MAP) and heart rate (HR) in unanesthetized freely moving ats, one day before the tests, rats were anesthetized again with .p. injection of ketamine (80 mg/kg of body wt) combined with ylazine (7 mg/kg of body wt) to receive a polyethylene tubing (PE- 0 connected to PE-50; Clay Adams, Parsippany, NJ, USA) inserted nto the abdominal aorta through the femoral artery. Another olyethylene tubing was also inserted into the femoral vein for Neurobiology 179 (2011) 227– 234 drug administration. Both cannulas were tunneled subcutaneously to the back of the rats to allow access in unrestrained, freely moving rats. We have evidence that the animals recovery from the anes- thesia and operative stress, because 1 day after the surgery the animals had normal drink and food intake and no impairment of motor activity. Although motor activity was not quantified, visual observation in their home cages and during handling revealed no apparent differences in reactivity or locomotion 1 day after the surgery. 2.2.2. Anesthetized animals General anesthesia was induced with 5% halothane in 100% oxygen. The rats received a tracheostomy and surgery was done under artificial ventilation with 1.4–1.5% halothane in 100% oxy- gen. All rats were subjected to the following previously described surgical procedures: femoral artery cannulation for arterial pres- sure measurement, femoral vein cannulation for administration of fluids and drugs, removal of the occipital bone and retracting the underlying dura mater for insertion of a pipette for microinjection into the medulla oblongata via a dorsal transcerebellar approach (Moreira et al., 2005, 2006). All animals were bilaterally vago- tomized to prevent any influence of artificial ventilation on phrenic nerve discharge (PND). The phrenic nerve was accessed by a dor- solateral approach after retraction of the right shoulder blade. In a group of rats (n = 7), used to test cardiorespiratory responses to hypercapnia, a complete baro- and peripheral chemoreceptor deafferentation was performed by sectioning the vagosympathetic trunks, the superior laryngeal nerves and the glossopharyngeal nerves (proximal to the junction with the carotid sinus nerves). Another rats (n = 6), used to test the cardiorespiratory responses to hypoxia, was a group of baro- and chemo-receptor intact rats, that had the vagi nerves carefully separated from the vagosympathetic trunk and selectively transected bilaterally. Splanchnic sympathetic nerve discharge (sSND) was recorded as previously described (Mandel and Schreihofer, 2008; Moreira et al., 2006; Takakura et al., 2011). The right splanchnic nerve was isolated via a retroperitoneal approach, and the segment distal to the suprarenal ganglion was placed on a pair of teflon-coated sil- ver wires that had been bared at the tip (250 �m bare diameter; A-M Systems, www.a-msystems.com). The nerves and wires were embedded in adhesive material (Kwik-Cast Sealant, WPI, USP), and the wound was closed around the exiting recording wires. Upon completion of the surgical procedures, halothane was replaced by urethane (1.2 g/kg of body weight) administered slowly i.v. All rats were ventilated with 100% oxygen throughout the experiment. The rectal temperature was maintained at 37 ◦C and the end tidal-CO2 (ETCO2) were monitored throughout the exper- iment with a capnometer (CWE, Inc., Ardmore, PA, USA) that was calibrated twice per experiment against a calibrated CO2/N2 mix. The adequacy of the anesthesia was monitored during a 20 min sta- bilization period by testing for the absence of withdrawal response, the lack of arterial pressure change and lack of change in the PND rate or amplitude to firm toe pinch. After these criteria were sat- isfied, the muscle relaxant pancuronium was administered at the initial dose of 1 mg/kg i.v. and the adequacy of anesthesia was there- after gauged solely by the lack of increase in arterial pressure and PND rate or amplitude to firm toe pinch. Approximately hourly sup- plements of one-third of the initial dose of urethane were needed to satisfy these criteria during the course of the recording period (4 h). 2.3. Intraparenchymal injections In the anesthetized rats placed in a stereotaxic frame (model 1760; David Kopf Instruments), muscimol (Sigma Chemicals Co., St-Louis, MO, USA, 2 mM or 100 pmol/50 nl, in sterile saline pH http://www.a-msystems.com/ logy & 7 s t r b i b l l i 2 2 t t c n A a M m b 1 r k t o t a T o s t f l m 2 s p t v e a E o D 2 ( c w 2 3 n i t C The statistical analysis was done with Sigma Stat version 3.0 (Jandel Corporation, Point Richmond, CA). The data are reported as M.T. Favero et al. / Respiratory Physio .4) was pressure injected into the commNTS (50 nl in 5 s) through ingle-barrel glass pipettes (20 �m tip diameter). Injections into he commNTS were made 400 �m caudal to the calamus scripto- ius, in the midline and 0.3–0.5 mm below the dorsal surface of the rainstem. In conscious freely moving rats, the same dose of muscimol was njected into the commNTS using 1 �l Hamilton syringes connected y polyethylene tubing (PE-10) to the injection needle 1.5 mm onger than the guide cannulas implanted into the brain. The solution of muscimol contained a 5% dilution of fluorescent atex microbeads (Lumafluor, New City, NY, USA) for later histolog- cal identification of the injection sites (Moreira et al., 2006). .4. Recordings of physiological variables .4.1. Conscious animals Twenty-four hours after the artery and vein cannulation, when he rats were completely recovered from the surgery and adapted o the environment of the recording room, the arterial catheter was onnected to a pressure transducer (MLT844, ADInstruments, Syd- ey, NSW, Australia) coupled to a preamplifier (Bridge Amp, ML221, DInstruments, Sydney, NSW, Australia) that was connected to Powerlab computer data acquisition system (PowerLab 16/30, L880, ADInstruments). The respiratory rate (fR, breaths/min) and the tidal volume (VT, l/kg) in conscious, freely moving rats were measured by whole- ody plethysmography as described in detail previously (Malan, 973; Onodera et al., 1997). All experiments were performed at oom temperature (24–26 ◦C). In brief, freely moving rats were ept in a plexiglass recording chamber (5 L) that was flushed con- inuously with a mixture of 79% nitrogen and 21% oxygen (unless therwise required by the protocol) at a rate of 1 L/min. The concen- rations of O2 and CO2 in the chamber were monitored on-line using fast-response O2/CO2 monitor (ADInstruments, NSW, Australia). he pressure signal was amplified, filtered, recorded, and analyzed ff-line using Powerlab software (Powerlab 16/30, ML880/P, ADIn- truments, NSW, Australia). The values of fR and VT analyzed were hose recorded for 2 min before the exposure to the stimulus and or 2 more min at the end of each stimulus, when breathing stabi- ized. Changes in the fR, VT, and minute ventilation (V̇E) (fR × VT; l/min/kg) were averaged and expressed as means ± SEM. .4.2. Anesthetized animals The mean arterial pressure, the discharge of the phrenic and planchnic nerves and the tracheal O2 and CO2 were recorded as reviously described (Moreira et al., 2006, 2007). Before starting the experiments, the ventilation was adjusted o have the ETCO2 at 3–4% at steady-state (60–80 cycles/s; tidal olume 1–1.2 ml/100 g). This condition was selected because 3–4% nd-expiratory CO2 was below the threshold of the PND. Variable mounts of pure CO2 were added to the breathing mixture to adjust TCO2 to the desired level. All analog data (ETCO2, sSND, PND and MAP) were stored n a computer via a micro1401 digitizer (Cambridge Electronic esign) and were processed off-line using version 6 of the Spike software (Cambridge Electronic Design) as described previously Takakura et al., 2006, 2011). The integrated phrenic nerve dis- harge (iPND) and the integrated splanchnic nerve discharge (iSND) ere obtained after the rectification and smoothing (� = 0.015 and s, respectively) of the original signal, which was acquired with a 0–300 Hz bandpass. Neural minute × volume (mvPND, a measure of the total phrenic erve discharge per unit of time) was determined by averaging the PND over 50 s and normalizing the result by assigning a value of 0 to he dependent variable recorded at the low levels of end-expiratory O2 (below threshold) and a value of 1 at the highest level of PCO2 Neurobiology 179 (2011) 227– 234 229 investigated (between 9.5 and 10%). The iSND was normalized for each animal by assigning the value of 100 to the resting SNA and the value of 0 to the minimum value recorded either during the admin- istration of a dose of phenylephrine that saturated the baroreflex (5 �g/kg, i.v.) or after the ganglionic blockade (hexamethonium; 10 mg/kg, i.v.). 2.5. Chemoreflex and baroreflex tests 2.5.1. Chemoreflex tests In anesthetized rats, the hypoxia was done by switching the breathing mixture from 100% O2 to 8–10% O2 balanced with N2 for 60 s, the same protocol used in a previous study (Takakura et al., 2006). The hypercapnia was done by increasing ETCO2 from 3–3.5% to 8–10% in hyperoxia condition (100% O2) for 5 min (Takakura et al., 2011). Conscious rats were maintained for at least 30 min at nor- moxia/normocapnia (21% O2, 79% N2, and <0.5% CO2) to adapt to the chamber environment before starting the measurements of the baseline arterial pressure and ventilation. Hypoxia was induced by lowering the O2 concentration in the inspired air down to a level of 8–10% for 60 s. Hypercapnia was produced by adding CO2 in the respiratory mixture up to 8–10% CO2 for 5 min under hyperoxic condition (90–92% O2), to minimize possible effects of peripheral chemoreflex activation (Trapp et al., 2008). 2.5.2. Baroreflex tests In conscious or anesthetized rats, the arterial baroreflex was examined by raising the arterial pressure with phenylephrine (5 �g/kg of body weight, i.v.) and lowering the arterial pressure with sodium nitroprusside (30 �g/kg of body weight, i.v). These doses of i.v. drugs were the same used in previous studies (Moreira et al., 2005, 2006; Takakura et al., 2009). For the i.v. injections, the drugs were prepared in sterile isotonic saline and the reflex tests were performed in the same order with drug injections separated by a 5 min interval. 2.6. Histology At the end of the experiments, rats were deeply anesthetized with halothane and perfused transcardially with saline followed by 10% buffered formalin (pH 7.4). The brain was removed and stored in the fixative for 24 h at 4 ◦C. The medulla was cut in 40 �m coro- nal sections with a vibrating microtome (Vibratome 1000S Plus – Starter CE, 220 V/60 Hz, USA), and stored in a cryoprotectant solu- tion at −20 ◦C (Nattie and Li, 2008). The injection site was verified with a conventional multifunction microscope (Olympus BX50F4, Japan). The section alignment between the brains was done relative to a reference section. To align the sections around NTS level, the mid-area postrema level was identified in each brain and assigned the level 13.8 mm (Bregma −13.8 mm) according to the atlas of Paxinos and Watson (1998). The coordinates of sections rostral and caudal of this reference section were calculated with respect to the reference section, using the number of intervening sections and the section thickness. 2.7. Statistics means ± standard error of the mean (SEM). The t-test or one way parametric ANOVA followed by the Newman–Keuls multiple com- parisons test were used as appropriate. The significance was set at p < 0.05. 230 M.T. Favero et al. / Respiratory Physiology & Fig. 1. (A) Photomicrograph showing the typical injection site into the commNTS in conscious rats (arrows). Scale = 1 mm. (B) The center of muscimol injections into the commNTS in the different rats tested represented on a single section (Bregma − I 3 3 4 B m t ( t t 3 c 3 i i ( ( o i a – b n p o p N r t b 14.3 mm, according to Paxinos and Watson, 1998). Scale = 1 mm. cc, central canal; O, inferior olive; py, pyramide; Sp5, spinal trigeminal tract; XII, hypoglossal nucleus. . Results .1. Histological analysis Muscimol injections into the commNTS were located about 00 �m caudal to the calamus scriptorius as illustrated in Fig. 1A and . A single injection of muscimol was administered in or near the idline as represented in Fig. 1B. Based on the area of the distribu- ion of the fluorescent microbeads, the injectate spread bilaterally approximately 500 �m from the injection center) and a little less in he rostrocaudal direction (approximately 300 �m from the injec- ion center). .2. Cardiorespiratory responses to hypoxia in anesthetized or onscious rats treated with muscimol into the commNTS .2.1. Anesthetized animals In urethane-anesthetized rats, in control conditions (after saline njected into the commNTS), a brief period of hypoxia (8–10% O2 n the breathing air for 60 s) produced an initial increase in MAP 26 ± 5 mmHg) in the first 5–10 s followed by a decrease in MAP −47 ± 6 mmHg) that reach the maximum at the end of the period f hypoxia (Fig. 2A1 and B1). In these conditions, hypoxia also ncreased sSND (283 ± 19% of the baseline) and mvPND (calculated s the product of phrenic nerve frequency and amplitude – f × a a measure of the total phrenic neural output) (149 ± 25% of the aseline) (Fig. 2A1, C and D). Injection of muscimol (100 pmol/50 nl) into the commNTS did ot change resting MAP (112 ± 3 mmHg, vs. saline: 110 ± 5 mmHg, > 0.05), sSND and mvPND (Fig. 2A2). The PND amplitude (98 ± 6% f control; p > 0.05) and duration (from 0.48 ± 0.02 to 0.47 ± 0.05 s, > 0.05) also did not change. Muscimol injected within the comm- TS blocked the pressor response (5 ± 2 mmHg, p < 0.01) and educed sympathoexcitation (93 ± 15% of the baseline, p < 0.01) and he increase in PND (20 ± 6% of the baseline, p < 0.01) produced y hypoxia (Fig. 2A2, 2B–D). Muscimol into the commNTS also Neurobiology 179 (2011) 227– 234 increased the hypotension produced by 60 s of hypoxia in anes- thetized rats (−63 ± 4 mmHg, p < 0.05) (Fig. 2A2 and B). 3.2.2. Conscious animals In conscious rats, in control conditions (after saline injected into the commNTS), 60 s of hypoxia (8–10% O2 in the inspired air) under normocapnia increased MAP (36 ± 3 mmHg), fR (60 ± 4 breaths/min), VT (4 ± 0.3 ml/kg) and V̇E (641 ± 28 ml/min/kg) and reduced HR (−96 ± 6 bpm) (Fig. 3A–E). Injection of muscimol (100 pmol/50 nl) into the commNTS, in conscious rats, did not change resting MAP (113 ± 6 mmHg, vs. saline: 117 ± 5 mmHg, p > 0.05) and HR (335 ± 21 bpm, vs. saline: 341 ± 18 bpm, p > 0.05). Muscimol injection within the commNTS reduced the increase on MAP (16 ± 2 mmHg, p < 0.05), fR (26 ± 3 breaths/min, p < 0.05), VT (1.8 ± 0.2 ml/kg, p < 0.05) and V̇E (250 ± 17 ml/kg/min, p < 0.01) and blocked the bradycardia (1 ± 2 bpm, p < 0.01) produced by hypoxia (Fig. 3A–F). 3.3. Cardiorespiratory responses to hypercapnia in anesthetized or conscious rats treated with muscimol injected into the commNTS 3.3.1. Anesthetized animals In urethane-anesthetized rats, in control conditions (after saline injected into the commNTS), hypercapnia (from 5% to 10% CO2 for 5 min) produced an immediate hypotension (−22 ± 4 mmHg) that was gradually reduced with MAP returning to or slightly above con- trol levels at the end of hypercapnia. Immediately after stopping hypercapnia (returning to 5% CO2), MAP increased (27 ± 5 mmHg) and returned to control values after around 5 min (Fig. 4A1 and B). In control condition, hypercapnia also increased sSND (108 ± 13% of baseline at 5% CO2) and mvPND (111 ± 8% of the baseline at 5% CO2) (Fig. 4A1, C and D). Injection of muscimol (100 pmol/50 nl) into the commNTS did not affect resting MAP, sSND or mvPND or hypercapnia-induced increase in MAP, sSND or mvPND in urethane anesthetized rats (Fig. 4A2, B–D). 3.3.2. Conscious animals In conscious rats, in control conditions (after saline injected into the commNTS), hypercapnia (8–10% CO2 in the inspired air) for 5 min under hyperoxic condition (92–98% O2, to minimize possible effects of peripheral chemoreflex activation) increased fR (55 ± 6 breaths/min), VT (3.7 ± 0.4 ml/kg) and V̇E (611 ± 19 ml/min/kg), however, produced no significant change in MAP (5 ± 2 mmHg) or HR (−4 ± 3 bpm) (Table 1). Injection of muscimol (100 pmol/50 nl) into the commNTS produced no change in resting MAP, HR and VE or on cardiorespiratory responses to hypercapnia in conscious rats (Table 1). 3.4. Cardiovascular responses to baroreflex activation in rats treated with muscimol injected into the commNTS 3.4.1. Anesthetized animals Injections of muscimol (100 pmol/50 nl) within the commNTS in anesthetized rats did not affect the pressor response and sympa- thoinhibition to i.v. phenylephrine (PHE, 5 �g/kg of body weight) or the hypotension and sympathoactivation to i.v injection of sodium nitroprusside (SNP, 30 �g/kg of body weight) (Table 2). PHE or SNP i.v. did not modify mvPND (Table 2). 3.4.2. Conscious animals In conscious rats, injection of muscimol (100 pmol/50 nl) within the commNTS also did not affect the pressor and bradycardic responses to i.v. PHE or the hypotension and tachycardia to i.v injec- tion of SNP (Table 3). Activation or deactivation of baroreceptors by M.T. Favero et al. / Respiratory Physiology & Neurobiology 179 (2011) 227– 234 231 Fig. 2. (A) Typical recordings of arterial pressure (AP), splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) in one rat representative of the group submitted to 60 s of hypoxia (8–10% O2) after injection of (A1) saline or (A2) muscimol (100 pmol/50 nl) into the commNTS under anesthesia. (B, C and D) Changes in mean arterial pressure (�MAP), sSND (�sSND) and mvPND (�mvPND), respectively, produced by 60 s of hypoxia after injection of saline or muscimol into the commNTS in anesthetized rats. * Different from saline (p < 0.05); n = 6 rats/group. Fig. 3. (A) Typical recordings of breathing, arterial pressure (AP), mean arterial pressure (MAP) and heart rate (HR) in one rat representative of the group submitted to 60 s of hypoxia (8–10% O2) after injection of saline or muscimol (100 pmol/50 nl) into the commNTS in conscious rats. (B–F) Changes in mean arterial pressure (MAP), heart rate (HR), ventilation (V̇E), respiratory frequency (fR) and tidal volume (VT), respectively, produced by hypoxia (8–10% O2) before (saline injection) and after muscimol injection into the commNTS. * Different from saline (p < 0.05); n = 6/group of rats. 232 M.T. Favero et al. / Respiratory Physiology & Neurobiology 179 (2011) 227– 234 Fig. 4. (A) Typical recordings of arterial pressure (AP), splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) in one rat representative of the group submitted to 5 min of hypercapnia (stepping ETCO2 from 5 to 10%) after (A1) saline or (A2) injection of muscimol (100 pmol/50 nl) into the commNTS under anesthesia. (B–D) Changes in mean arterial pressure (�MAP), sSND (�sSND) and mvPND (�mvPND), respectively, produced by hypercapnia after injection of saline or muscimol into the commNTS in anesthetized rats. * Different from saline (p < 0.05); n = 6 rats/group. Table 1 Cardiorespiratory responses to hypercapnia in conscious rats treated with muscimol injected into the commNTS. Treatment n �MAP (mmHg) �HR (bpm) �fR (breaths/min) �VT (ml/kg) �V̇E (ml/min/kg) Saline (control) 6 +5 ± 2 −4 ± 3 +55 ± 6 +3.7 ± 0.4 +611 ± 19 Muscimol 6 +5 ± 3 −6 ± 2 +53 ± 5 +3.8 ± 0.5 +668 ± 28 Values are means ± SEM. n = number of rats. �MAP, changes in mean arterial pressure; �HR, changes in heart rate; �fR, changes in respiratory frequency; �VT, changes in tidal volume; �V̇E , changes in ventilation. Muscimol (100 pmol/50 nl). Hypercapnia (8–10% CO2 in the inspired air) for 5 min under hyperoxic condition (100% O2). Table 2 Cardiovascular responses to phenylephrine and sodium nitroprusside in anesthetized rats treated with muscimol injected into the commNTS. Treatment n �MAP (mmHg) �sSND (%) �mvPND (%) PHE SNP PHE SNP PHE SNP Saline (control) 5 +32 ± 3 −34 ± 5 −98 ± 3 +101 ± 6 +9 ± 9 +6 ± 13 Muscimol 6 +34 ± 7 −38 ± 4 −94 ± 8 +97 ± 13 +10 ± 4 +8 ± 11 V eigh a vPND, P ( 3 n ( p w r n ( b alues are means ± SEM. n = number of rats. PHE, phenylephrine (5 �g/kg of body w rterial pressure; �sSND, changes in splanchnic sympathetic nerve discharge; �m HE and SNP i.v., respectively, did not change V̇E in conscious rats Table 3). .5. Specificity of commNTS as the site for the effects of muscimol Injections of muscimol outside the commNTS (n = 4) did ot change the pressor (25 ± 4 mmHg, p > 0.05), sympathetic 270 ± 15% of baseline, p > 0.05) and phrenic (136 ± 9% of baseline, > 0.05) responses evoked by peripheral chemoreflex activation ith brief period of hypoxia in anesthetized rats. In conscious ats, the injection of muscimol outside commNTS (n = 7) produced o change on pressor (33 ± 6 mmHg), fR (54 ± 9 breaths/min), VT 4.2 ± 0.4 ml/kg) and V̇E (631 ± 33 ml/min/kg) responses and on the radycardia (−84 ± 11 bpm) produced by hypoxia. t); SNP, sodium nitroprusside (30 �g/kg of body weight); �MAP, changes in mean changes in phrenic nerve discharge. 4. Discussion The present results provide functional evidence that the caudal portion of the commNTS is essential for the pressor response and the increase in the SND and breathing produced by hypoxia in con- scious or anesthetized rats. However, the results show no evidence that this portion of the NTS is involved in mediating cardiorespi- ratory responses to hypercapnia. In addition, the inhibition of the caudal commNTS neurons did not modify the responses produced by baroreflex activation as previously demonstrated (Moreira et al., 2009). The changes in arterial pressure produced by hypoxia or hyper- capnia are the result of two opposite effects, a vasodilation due to the peripheral effect of the changes in O2 or CO2 and the M.T. Favero et al. / Respiratory Physiology & Neurobiology 179 (2011) 227– 234 233 Table 3 Cardiovascular responses to phenylephrine and sodium nitroprusside in conscious rats treated with muscimol injected into the commNTS. Treatment n �MAP (mmHg) �HR (bpm) �V̇E (ml/kg/min) PHE SNP PHE SNP PHE SNP Saline (control) 6 +49 ± 5 −28 ± 6 −59 ± 8 +88 ± 4 +21 ± 8 +11 ± 5 Muscimol 6 +54 ± 6 −31 ± 5 −44 ± 11 +93 ± 8 +25 ± 7 +18 ± 8 V eigh a c t t a M c t b o a B l a i d p t h a e o w i p r s n c s a c r r c t p t t m t N c e s p p p i c c s alues are means ± SEM. n = number of rats. PHE, phenylephrine (5 �g/kg of body w rterial pressure; �HR, changes in heart rate; �V̇E , changes in ventilation. entrally mediated vasoconstriction that depends on chemorecep- or and sympathetic activation. Previous studies have suggested hat anesthetics may affect neurotransmission on the brainstem nd consequently reflex responses (Accorsi-Mendonç a et al., 2007; achado and Bonagamba, 1992). Therefore, in the present study, ardiorespiratory responses to hypoxia and hypercapnia were ested in anesthetized rats and also in conscious rats using whole- ody plethysmography, which allows simultaneous measurements f respiratory and cardiovascular responses without restraining the nimal (Biancardi et al., 2008; Braccialli et al., 2008; de Paula and ranco, 2005). In urethane-anesthetized, vagotomized and artificially venti- ated rats, in control conditions, hypoxia or hypercapnia produced dual response on arterial pressure. The hypoxia produced an nitial increase in MAP in the first 5–10 s that was followed by a ecrease in MAP that reach the minimum value at the end of the eriod of hypoxia. The hypercapnia reduced arterial pressure in he first minute followed by an increase at the end of the 5-min ypercapnia. The hypoxia or hypercapnia rapidly increased PND nd gradually increased sSND which reaches the maximum at the nd of the test. In conscious rats, in control conditions, hypoxia r hypercapnia increased ventilation and hypoxia increased MAP, hereas hypercapnia produced no change in MAP. The blockade of neuronal activity with muscimol injection nto the commNTS almost abolished the pressor, sympathetic and hrenic responses to hypoxia in anesthetized rats and partially educed the pressor and respiratory responses to hypoxia in con- cious rats, whereas the same treatment in the commNTS produced o changes in the cardiorespiratory responses to hypercapnia in onscious or anesthetized rats. Therefore, in anesthetized or con- cious rats, it seems that chemoreflex-mediated cardiovascular nd respiratory responses to hypoxia are strongly dependent on audal commNTS mechanisms. However, in conscious rats, neu- onal blockade in the commNTS with muscimol only partially educed cardiorespiratory responses to hypoxia. The effects of mus- imol injected into the commNTS in conscious rats are similar to hose previously demonstrated in the working heart-brainstem reparation after combining glutamatergic and purinergic recep- or blockade in the commNTS (Braga et al., 2007), which suggest hat in this case cardiorespiratory responses to hypoxia are also ediated by signals that arise from other central sites not related o commNTS. A previous study showed that electrolytic lesions of the comm- TS abolished the pressor and bradycardic responses to peripheral hemoreceptor activation with i.v. injection of KCN (Colombari t al., 1996). It is interesting to note that the results of the present tudy showed that muscimol into the commNTS only reduced the ressor responses to hypoxia in conscious rats, whereas in the revious study electrolytic lesions of the commNTS abolished the ressor response to i.v. KCN. These differences also suggest that, n conscious rats, besides the activation of peripheral chemore- eptors, additional mechanisms are activated by hypoxia, probably entrally, that do not depend on commNTS (Colombari et al., 1996). Although the cardiorespiratory responses to hypoxia are trongly dependent on commNTS neuron activation, the present t); SNP, sodium nitroprusside (30 �g/kg of body weight); �MAP, changes in mean results suggest that the same commNTS neurons involved in the responses to hypoxia are not involved in the cardiorespiratory responses to hypercapnia. Muscimol injections into the commNTS did not change the increase in arterial pressure, SND or breath- ing produced by hypercapnia. However, a previous study showed that it is possible to reduce the respiratory responses to hypercap- nia by muscimol microdialysis in the commNTS, suggesting that commNTS may detect CO2 (Nattie and Li, 2008). The same study also showed that muscimol microdialysis in the commNTS did not affect respiratory responses to hypoxia when rats were tested at room temperature of 24 ◦C, the same room temperature that rats were exposed in the present study. Therefore, the present and the previous study show different effects of the commNTS inhi- bition with muscimol in the control of the respiratory responses to hypoxia or hypercapnia. Possible reasons for the different results are the differences in the site of microdialysis/injections into the commNTS, the volume of microdialyis/injections and the concen- tration of muscimol released in the commNTS. In the previous study (Nattie and Li, 2008), microdialysis probes released muscimol into the commNTS bilaterally at the level of the area postrema, whereas in the present study just one injection was performed in the mid- line around 400 �m caudal to the area postrema, i.e., the previous study tested the effects of muscimol in a more rostral portion of the commNTS and the present study in a more caudal portion of the commNTS. Although, different sites of injections/microdialysis seem to be the main reason for the different results, in the pre- vious study, the concentration of muscimol was 0.5 mM and the volume of microdialyis was 4 �l/min continuously throughout the entire experiment (Nattie and Li, 2008), whereas in the present study the concentration of muscimol was 2 mM and a volume of 50 nl was injected in a single injection. Although in both studies the nomenclature is the same (commissural NTS), they did not test the same area/neurons: the present study tested a more caudal por- tion of the commNTS and the previous study (Nattie and Li, 2008) tested a more rostral part of the commNTS. Therefore, based on the present and the previous study (Nattie and Li, 2008) it is possi- ble to suggest that different parts of the commNTS are involved in the respiratory responses to hypoxia and hypercapnia. According to the present results, a more caudal portion of the commNTS is involved in cardiorespiratory responses to hypoxia, whereas a pre- vious study suggests that a more rostral portion of the commNTS is the site of the pH-sensitive cells of the NTS important mainly for the respiratory responses to hypercapnia. 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Chemosensory control by commissural nucleus of the solitary tract in rats 1 Introduction 2 Methods 2.1 Animals 2.2 Surgical procedures 2.2.1 Conscious animals 2.2.2 Anesthetized animals 2.3 Intraparenchymal injections 2.4 Recordings of physiological variables 2.4.1 Conscious animals 2.4.2 Anesthetized animals 2.5 Chemoreflex and baroreflex tests 2.5.1 Chemoreflex tests 2.5.2 Baroreflex tests 2.6 Histology 2.7 Statistics 3 Results 3.1 Histological analysis 3.2 Cardiorespiratory responses to hypoxia in anesthetized or conscious rats treated with muscimol into the commNTS 3.2.1 Anesthetized animals 3.2.2 Conscious animals 3.3 Cardiorespiratory responses to hypercapnia in anesthetized or conscious rats treated with muscimol injected into the c... 3.3.1 Anesthetized animals 3.3.2 Conscious animals 3.4 Cardiovascular responses to baroreflex activation in rats treated with muscimol injected into the commNTS 3.4.1 Anesthetized animals 3.4.2 Conscious animals 3.5 Specificity of commNTS as the site for the effects of muscimol 4 Discussion Acknowledgements References